Project Summary/Abstract Impaired fertility due to compromised ovarian function affects millions of American women today. The ovarian follicle (each contains one oocyte) is the fundamental functional tissue unit of the ovary. Therefore, isolation and cryopreservation of ovarian follicles for in vitro culture to obtain healthy fertilizable oocytes have been regarded as a promising strategy for restoring and preserving female fertility. However, none of the methods used today for follicle culture recapitulate the mechanical heterogeneity experienced by both the primary and pre-antral follicles in the ovary. Using a non-planar microfluidic device, we have recently fabricated a biomimetic ovarian microtissue that consists of a more rigid alginate hydrogel shell and a softer collagen core to mimic the harder ovarian cortex and softer ovarian medulla, respectively. The follicle is partially embedded both in the core and shell, which recapitulates the mechanical heterogeneity experienced by the follicles in vivo. With this biomimetic ovarian microtissue, we revealed that the mechanical heterogeneity is crucial for developing early pre-antral follicles to the antral stage and ovulation to release oocytes. We hypothesize that the biomimetic ovarian microtissue system can be further developed for in vitro culture of both primary and early pre-antral follicles. For follicle cryopreservation, contemporary approaches require either a highly toxic concentration (up to ~8 M) of membrane-penetrating cryoprotectants (CPAs) to vitrify (i.e., cooling to cryogenic temperature without ice formation) the follicles, or slowly freezing the follicles to form extracellular ice and dehydrate them. The latter is associated with inevitable physicochemical damage to cells due to ice formation. Our recent studies revealed that alginate hydrogel microencapsulation is exceptional in suppressing ice formation and growth, which allows vitrification of a variety of stem cells at a low CPA concentration (1.5-2 M) with high viability and intact function post cryopreservation. This low-CPA vitrification approach combines all the advantages of the conventional approaches for cell cryopreservation while avoiding their shortcomings. We hypothesize that this low-CPA vitrification approach can be used to cryopreserve the primary and early pre-antral follicles encapsulated in the biomimetic microtissue, due to the presence of an alginate hydrogel shell in the microtissue. The objective of this project is to test the aforementioned two hypotheses with three specific aims: 1), to develop a computational model for understanding the complex multi-phase flow occurred during microfluidic encapsulation of follicles in the biomimetic ovarian microtissue; 2), to microencapsulate both primary and early pre-antral follicles for biomimetic 3D culture in vitro; and 3), to cryopreserve primary and early pre-antral follicles encapsulated in the biomimetic ovarian microtissue by low-CPA vitrification. The novel non-planar microfluidic device, biomimetic ovarian microtissue system, and low-CPA vitrification technology are valuable for studying follicle biology, screening pharmaceutical drugs, and restoring/preserving the fertility of women.